The K562 cell is being studied as a model of human globin gene expression. K562 cells express embryonic and fetal hemoglobins but no hemoglobin A, Beta-globin chain, nor Beta-globin mRNA. Understanding the pattern of globin expression in K562 cells may yield insights into globin expression in normal cells. The failure to express Beta-globin mRNA could result from an acquired mutation in the Beta gene or from repression of the Beta-globin locus. In the present study, we examined the structure of the K562 Beta-globin gene by 1) restriction mapping of genomic K562 DNA; 2) cloning the K562 Beta gene; 3) and studying expression of the cloned gene introduced into an heterologous cell. Our restriction mapping of genomic K562 DNA demonstrates that the Beta-globin gene has sustained no major insertions, deletions, or rearrangements. Furthermore, our analysis of a polymorphic site for the restriction endonuclease Ava II demonstrates the presence of two different alleles at the Beta locus. Therefore, to abolish Beta globin expression a mutation at the Beta-globin locus must be small and must affect multiple loci. We have recently cloned the K562 Beta-globin gene for expression studies. By restriction enzyme analyses our cloned Beta-globin gene is structurally normal. The cloned gene was introduced into an heterologous expression system and transcription products were analyzed. The cloned Beta-globin gene was subcloned into pLTN3B, an expression vector with enhancers for eukaryotic gene transcription; the constructed plasmid was used to transform COS cells; and the COS cell RNA was analyzed by S1 mapping for Beta-globin message. In COS cells the K562 Beta-globin gene is transcribed; and the message is appropriately initiated, processed, and terminated. These results suggest that a mutation is unlikely to be responsible for the absence of Beta-globin expression; but rather that the lack of Beta-globin transcription in the K562 cell results from the regulatory factor environment within the cell. We have developed methods for introducing cloned genes into K562 cells to study directly human erythroid regulatory factors and the DNA sequences required by such factors.